Genetic Control of Nortriptyline Kinetics in Man A Study of Relatives of Propositi with High Plasma Concentrations*

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1 Journal o Medical Genetics (1971). 8, 19. Genetic Control o Nortriptyline Kinetics in Man A Study o Relatives o Propositi with High Plasa Concentrations* MARIE ASBERG, DAVID A. PRICE EVANS, and FOLKE SJOQVIST Fro the Departent o Psychiatry, Karolinska Sjukhuset, Stockhol, Sweden, the University o Liverpool, England, and the University o Link6ping, Sweden Considerable variability has been observed in the steady-state plasa concentrations o nortriptyline in psychiatric patients undergoing treatent with the drug (Haer, Idestro, and Sjoqvist, 1967). Soe patients have been ound to have exceptionally high plasa levels oring a ode which was separated ro the great ajority (Sj6qvist et al, 1968). Nortriptyline (NT) steady-state plasa levels have been shown to be under genetic control (Alexanderson, Price Evans, and Sjoqvist, 1969) in a study o identical and raternal twins receiving a sall daily dose o the drug. The study o Alexanderson et al (1969) did not appear, however, to contain persons who developed exceptionally high steady-state plasa levels. Nortriptyline is easily absorbed ro the intestine (Haer and Sjoqvist, 1967), individual dierences in the binding o plasa protein are negligible (Borga et al, 1969), and the renal excretion o unchanged nortriptyline accounts or less than 500 o the daily dose under steady-state conditions (Sjoqvist et al, 1968). The variability in steady-state plasa concentration is thereore presuably due to dierences in rate o etabolis and apparent volue o distribution (Alexanderson et al, 1969). Investigations in huan volunteers suggest that rate o etabolis is the quantitatively ost iportant source o variance (B. Alexanderson and 0. Borga, unpublished observations). The objects o the present study were to attept to ascertain whether patients achieving very high steady-state NT plasa concentrations do so because o their genetic constitution and whether the anner o genetic control was onogenic or polygenic. Received 1 October 19. * This study was supported by grants ro the Swedish Medical Research Council (B-61P , K69-1X-9-GO1 and B-1x ), the National Institutes o Health, Bethesda USA (GM ), the Meorial Fund o Proessor Bo Gadelius, and the Karolinska Institute. I- T.M.G. 19 Material and Methods High Nortriptyline Plasa Level Propositi. Three 'high-level' patient propositi had been ascertained. Two o these (L.S. and H.I.) were retested or plasa NT concentration using the technique described below. The third proposita (L.I.) was not available or retesting. The indings in the propositi are suarized in Table I. Relatives available or Nortriptyline Testing. Pedigrees were available via propositi L.S. and H.I. and are shown in Figure 1. For the third proposita (L.I.) only two sisters were available. In total 9 relatives were tested. Rando Subjects. Twenty rando subjects were tested. These coprised 4 unrelated spouses o relatives, 3 hospital eployees such as doctors and nurses, 6 students, 4 patients ree o organic disease, and 3 healthy relatives o 3 psychiatric patients whose nortriptyline or desethyliipraine steady-state plasa levels were in the ain ode o Fig. 8 o Sjoqvist et al (1968). Dosage o Nortriptyline and the Taking o Blood Speciens. All subjects were given NT as sugar coated coercial tablets (Sensaval, Pharacia). The dose was g three ties daily or all subjects and was given or up to 8 days. Blood speciens were generally taken on days 4, 5, 6, and 8, but soeties they had to be drawn on dierent days. Three subjects (one relative and two rando subjects) stopped taking the drug within three days due to side eects. Two subjects (one relative and one rando subject) who had side eects were unable to continue the drug ater day 6. Clotting o the blood speciens was prevented with heparin and 05 l 0-1-N hydrochloric acid was added to each -0 l o plasa as a preservative. The speciens were stored at -0 C until deterinations o plasa NT concentrations were perored, which generally occurred within weeks. Deterination o Plasa Nortriptyline Concentrations. Plasa concentrations o NT were deterined by the ethod described by Haer and J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

2 130 A Asberg, Evans, and Sj6qvist TABLE I 'HIGH-LEVEL' PROPOSITI Deterination o steady-state plasa concentration o nortriptyline ~~~~~~Steady-state Dosage o Data Data derived ro Subjectt Sx Sex Weight Do o Concoitant (kg) (gntid.) Drug Treatent Plasa Concentration (g t.i.d ~~~~o NT (MAg/l) Sjoqvist et al, 1969 L.S. Feale Pentobarbital (0-1 g) Haer et al, 1967 L.I. Feale Methaqualone (0 g) 138 Sjoqvist et al, 1968 H.I. Feale Oral iron and vitains 75 B Data obtained on retesting in present study (Subject L.I. was not available or retesting. Figures within brackets indicate values obtained in connection with the study o Haer et al, 1967) Plasa Concentration o NT on g t.i.d. Subject (tsg/l) Coents Day 4 Day 5 Day 6 Day 8 Day 10 L.S Interval between irst and second test 1 year. No concoitant drug treatent. Free ro exposure to other drugs onths beore testing L.I. (100) (17) (1) H.I Interval between irst and second test two years. The patient had been receiving phenothiazines continuously in the eantie, and her weight on retesting was 83 kg. Free ro other drugs weeks beore retesting Brodie (1967) and urther described by Sj6qvist et al (1969). Unknowns were processed in duplicate, and standards in triplicate at 30, 50, 100, and 00 Hg NT per l were processed in each deterination procedure. Results Full details o the results are shown in Table II. Exclusions o Subjects because o Concoitant Disease and Drug Therapy. Nine subjects were either diseased and/or receiving concoitant drug therapy. Details are given in Table III together with the arbitrary decision on inclusion in urther calculations. Subjects receiving barbiturates or contraceptive steroids were excluded. Pedigree A (L.S.) e@qtq~~4 5U96 7 %9t Pedigree I B (H.-I.) )Nt4X4) 1 ()( -proposita $=not exained FIG. 1. Pedigrees or two propositi with exceptionally high steadystate plasa levels o nortriptyline. Barbiturates are known to inluence the steady-state plasa levels o tricyclic antidepressants (Haer et al, 1967), and studies in progress by von Bahr et al (unpublished data ro this laboratory) indicate that contraceptive steroids aect the kinetics o other drugs, such as antipyrine. All other 40 subjects were ree o signiicant disease and had not been exposed to drugs or other cheicals during at least 3 onths beore the study. Speciic enquiries were ade concerning all doctor's prescriptions, patent edicines, laxatives, vitains, tonics, and contraceptive steroids. The Use o Plasa Concentrations to Characterize an Individual. Two easureents were utilized. First, the plasa NT concentrations on the sixth day; these were available or all subjects. Second, the area under the curve described by plotting NT concentrations (y) against days o NT therapy (x). The advantage o the latter procedure was that it utilized all observations available. One subject (Table II, case 3) could not be used or this procedure because o the paucity o the data available. A diiculty which arose with soe subjects was the absence o a plasa NT concentration result on the eighth day, and this was resolved as ollows. When the plasa NT concentration appeared to be approaching steady state on day 6, the ean o the values o days 4, 5, and 6 was used as the value or day 8. When the plasa NT concentration appeared to be increasing on day 6, J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

3 Genetic Control o Nortriptyline Kinetics in Man TABLE II BASIC DATA ON SUBJECTS STUDIED Subject No. Designation Sex Weight Age Plasa Nortriptyline Concentration, gg/l on day inpedigree (g A:I:1 A:II:1 A:II:3 A:II:5 A:II:7 A:II:8 A:II :10 A:II:ll A:II:1 A.III.1 A:III: A:III:3 A:III:4 A:III:5 A:III:6 A:III:7 A:III:8 A:III :9 A:III:10 A:III:ll B:I:1 B:I: B:II:1 B:II: B:II:3 B:II:4 B:II:6 A:II: A:II:4 A:II:6 AJII: the day 6 value was used as the day 8 value. The or 18 eales 1-93 (SE 0-04); t to copare eans upper boundaries o the areas were obtained by was Hence there is no signiicant inluence o joining the concentration values with straight lines. sex on the easureent. The eans and standard deviations or relatives Plasa Nortriptyline Concentrations on and rando subjects are shown in Table V. Both Day 6. Log1o transorations o these values were the ean and the variance are signiicantly greater approxiately norally distributed. There was a in all the relatives o high-level propositi than in all signiicant regression o the log1o, 6th-day plasa the rando subjects. NT concentration on body weight (Table IV, row 1) and consequently the values were adjusted to a Areas under Nortriptyline-day Curves. standard weight o 68 kg using the calculated re- The distribution o these values was skewed and gression coeicient. There was no signiicant could be rendered approxiately noral by log1o regression o weight-adjusted log1o, 6th-day plasa transoration. A signiicant regression was ound NT concentrations on age (Table IV, row ). o log1o area on body weight (Table IV, row 3). The ean or 6 ales was 1 90 (SE 0 04) and Hence log1o area values were adjusted to a standard J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

4 13 Asberg, Evans, and Sj6qvist TABLE III INDIVIDUALS WITH DISEASE AND ON CONCOMITANT DRUG THERAPY Subject Designation Alleged Disorder Concoitant Drug Therapy Included in No. in Pedigree Coputations 6 A :I:8 Gout Phenylbutazone or days beore NT Yes test 11 A:III: Nil Contraceptive steroids No 17 AJII:18 Nil Contraceptive steroids No 19 A :111:10 Nil Contraceptive steroids No 1 B:1:1 Convalescing ater surgery Oral iron and olic acid Yes or siple oesophageal obstruction B:I: Cholecystitis Papaverine 0-8 g t.i.d. and a tetra- Yes cycline (Tetralysal Erco) 150 g 4 ties daily 8 Rheuatoid arthritis Digoxin 0- g t.i.d. and acetyl sali- Yes cylic acid 34 g three ties daily 33 AJ:I:8 'Run down' Cobination tablet (pentobarbital 50 No g and eprobaate 00 g) nightly 43 Nil Contraceptive steroids No TABLE IV SOME REGRESSIONS COMPUTED FROM THE BASIC DATA Row nuber x y n b a r t p 1 Body weight (kg) Logl0 6th day plasa NT < concentration Age (yr) Ditto, adjusted or body NS weight 3 Body weight (kg) Log10 NT concentration < 0-01 day area 4 Age (yr) Ditto, adjusted or body NS weight n - nuber o pairs o observations b regression coeicient a = intercept (ie, value o y when x = 0 in units o y) r correlation coeicient t= to test or signiicance o b p signiicance level NS = not signiicant at 0 05 level body weight o 68 kg using the calculated regression coeicient. There was no signiicant regression o weight-adjusted logi0 area on age (Table IV, row 4). The ean or ales was - (SE 0-04) and or 18 eales - (SE 0O05); t to copare eans was 0-3. Hence there is no signiicant inluence o sex on this easureent. The eans and standard deviations or rando subjects and relatives are shown in Table V. The ean or all the relatives o high-level propositi is signiicantly higher than that or all the rando subjects. Further Considerations o Results within Failies. The two easureents o NT etabolis used were (1) log1o 6th-day plasa NT concentrations adjusted or body weight and () loglo area under plasa NT concentrations curve adjusted or body weight. They give very siilar results as can be seen in Figs. and 3, and Table V. Pedigree A, Fig. 1, gives urther relevant inoration. Full sibs o L.S. lie towards the upper end o the distribution curve or rando subjects, while their spouses lie towards the centre o the sae distribution. Ospring o the ull sibs o L.S. give or easureent (1) a ean o 1 95 (SD 0 6), and or easureent () a ean o -73 (SD 0 0). Discussion Coparison o Results in Rando Subjects and Relatives o High-level Propositi. The results o the high-level propositi were approxiately at the upper tail o the distribution curve o their relatives and o the distribution curve o the rando subjects. This observation in itsel suggested that a polyorphis (ie, variability controlled ainly by alleles at a single locus) was not present. Hence a polygenic control o the easureent under discussion seeed ore likely. J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

5 TABLE V SUMMARIZED RESULTS ON VARIOUS CATEGORIES OF SUBJECTS Genetic Control o Nortriptyline Kinetics in Man Measureent Log6t-day lasa Log area under Categry Category o Log NT6th-day concentration, plasa plasa tration NT curve, concen- Subjects adjusted or body adjted or body weight (C) ~weight (A) n Mean SD n Mean SD Rando subjects All relatives o high level propositi 6 1* Ospring o ull siblings o L.S COMPARISONS OF MEASUREMENTS C AND A BETWEEN CATEGORIES OF SUBJECTS Categories Copared t DF p Means Rando subjects, C vs all relatives o C -1 4 < 0 05 Rando subjects, A vs all relatives o A < 0-01 Rando subjects, C vs ospring o ull sibs o L.S., C NS Rando subjects, A vs ospring o ull sibs o L.S., A < 0-05 Variances Rando subjects, C vs all relatives o C ; 6 < 0-05 Rando subjects, A vs all relatives o A ; NS Rando subjects, C vs ospring o ull sibs o L.S., C ; 7 < 0 05 Rando subjects, A vs ospring o ull sibs o L.S., A ; 7 NS n = nuber o observations SD = standard deviation DF = degrees o reedo Lack o Eect o Age on Plasa Nortriptyline Concentrations. The incidence o adverse reactions in general rises with increasing age, and this observation reains valid when digitalis reactions are deducted (Hurwitz, 1969). This increased susceptibility o the elderly to adverse eects could theoretically be due to a diinished capacity to eliinate or etabolize drugs or to increased susceptibility o 'receptors'. The present work gives no evidence o ajor alterations in the kinetics o NT with increasing age. Association o Side Eects with Plasa Concentration o Nortriptyline. The healthy relatives and rando subjects tested in this study were given an explanation o the possible adverse eects o NT when their cooperation was obtained beore starting drug therapy. During drug therapy and 0 4. rando subjects v 11 ull sibs o L.S. Z spouses o ull sibs o L.S '0 ospring o ull sibs o L.S '0 all relattives o high-level propositi :J I Log,0 plasa NT concentration on 6th day (adjusted or body weight) FIG.. Distributions o logl0 plasa nortriptyline on the 6th day, adjusted or body weight, or various categories o subjects. 4-1A 4 l z41-4 relatives I._.5 '0 30 rando subjects I ' 0 - I 1,,~~~. R., ull sibs - -5, Log,0 area under plasa NT concentration curve (adjusted or body weight) FIG. 3. Distributions o log10 area under plasa nortriptyline concentration curve, adjusted or body weight, or various categories o subjects... a beore the analyses o plasa NT concentrations, a record was ade o whether they volunteered coplaints o side eects. Individuals have been categorized as to whether the value o plasa NT concentration on day 6 was larger or saller than the edian o the 44 subjects exained. A test or the association o adverse eects and plasa NT concentration is shown in Table VI and is signiicant. The exact probability is or one tail and or two tails. Since the biological situation suggests that persons with low plasa levels are not likely to have ore side eects than persons with high plasa levels, the one tailed test can be _ J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

6 134 TABLE VI ASSOCIATION OF SIDE EFFECTS AND PLASMA CONCENTRATION OF NORTRIPTYLINE Plasa NT Concentration Side Eects Below Above Total Population Population Median Median Not volunteered Volunteered Total 44 x=4 7 considered appropriate. The inding is in agreeent with recent observations in depressed patients treated with nortriptyline (Asberg et al, 19). The Nature o the Genetic Control o Nortriptyline Plasa Concentrations. There are two ain explanations to be considered. (1) That NT plasa levels are controlled as a polyorphis with 'high-level' propositi being genetically constituted as rare recessives, and () that NT plasa levels are controlled by a polygenic syste with 'high-level' propositi being selected ro the high end o the distribution curve o the population. In avour o the irst explanation was the inding o a separate ode o high-level propositi in the histogra o Sjoqvist et al (1968). It ust be realized, however, that this distribution is skewed and had not been noralized by log1o transoration. In addition, the analysis o the data is coplicated by the act that the subjects under study were patients and had received various types o psychotropic drugs, including barbiturates, beore the study. In avour o the second explanation are the ollowing: (a) both ean and variance o log1o sixthday plasa NT concentrations and ean o log1o areas under NT plasa concentration curves are larger in relatives o high-level propositi than in rando subjects. (b) The ull sibs o high-level propositi studied did not appear to all into two odes. However, since only 1 ull sibs were studied it could be (on the onogenic hypothesis) that only one other recessive phenotype was ound; the probability o this occuring is (c) The ospring o ull sibs o high-level propositi do not appear to or two separate odes. Their ean log1o area under plasa NT concentration curve and the variance o log1o 6th-day plasa NT concentration are larger than those ound in rando subjects. There sees to be little deinitive evidence against the second explanation. It would thereore appear very probable that NT kinetics in an Asberg, Evans, and Sjoqvist is under polygenic control. Fro the evidence available it is not possible to say either how any loci, or how any alleles at a given locus exercise genetic control o NT plasa concentrations. The results o Alexanderson et al (1969) in non-drug treated raternal twins indicate considerable intrapair dierences in only 4 out o the 11 twinships studied. This ight hint that the nuber o genes involved in the control o NT plasa concentrations is sall. Suary Twenty-nine relatives o 3 patient propositi developing extreely high plasa concentrations o nortriptyline and 0 rando subjects were given g nortriptyline orally three ties daily during 8 days. Plasa concentrations o nortriptyline were deterined on days 4, 5, 6, and 8. Forty o the subjects were healthy and had not been exposed to drugs or cheicals 3 onths beore the study. O the reaining 9 subjects, 5 were excluded ro all urther calculations because they had received barbiturates or contraceptive steroids. Two easureents were used to characterize individuals: (1) plasa concentration o nortriptyline on the sixth day o treatent and () the area under the curve obtained by plotting nortriptyline concentrations (y) against days o treatent (x). The distributions o both were skewed but could be rendered approxiately noral by log1o transoration. There was a signiicant regression o both on body weight, hence all values were adjusted to a standard body weight. There was no correlation between weight-adjusted values and age, nor any dierence between ales and eales. There was a signiicant association between the occurrence o side eects and a plasa concentration above the population edian. There was no tendency towards biodality in the distributions o all subjects, all relatives or all ull siblings o high-level propositi on either o the easureents. The variances were larger in relatives o high-level propositi than in rando subjects, and the ean o estiate () was larger in the relatives than in the rando subjects. Spouses and ospring o the ull sibs o one o the propositi were tested. While the values o the ull sibs on both easureents were at the upper tail o the distribution o the rando subjects, the spouses were in the centre o the distribution. In the ospring o the ull sibs, the variance o easureent (1) and the ean o easureent () were larger than in the rando subjects. It is concluded that the anner o inheritance o nortriptyline kinetics is likely to be polygenic. J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by

7 The authors wish to thank the propositi, relatives, and rando subjects or their ost helpul cooperation. They would also like to thank Dr Daniel Perers (Visby), Proessor Jan-Otto Ottosson (Uea), Proessor Lars Magnus Gunne (Uppsala), Dr Olle Spangberg (Kristinehan), Dr Marten Kalling (Visteras), and Dr L. Tigerstedt (Anaset); and Proessor P. M. Sheppard (University o Liverpool) or his advice concerning the genetic analyses. Thanks are also due to the University o Liverpool or allowing D.A.P.E. leave o absence to work on the project at the Karolinska Institute, Stockhol; and Geigy Pharaceuticals Ltd (UK) or generously providing travel and subsistence grants to enable hi to participate in research at the Karolinska Institute. RaRsNCES Alexanderson, B., Price Evans, D. A., and Sjoqvist, F. (1969). Steady-state plasa levels o nortriptyline in twins. Inluence o g:netic actors and drug therapy. British MedicalJournal, 4, Asberg, M., Cronhol, B., Sjoqvist, F., and Tuck, D. (19). The Genetic Control o Nortriptyline Kinetics in Man 135 correlation o subjective side-eects with plasa concentrations o nortriptyline. British MedicalJournal, 4, Borgi, O., Azarno, D. L., Ply Forsell, G., and Sjoqvist, F. (1969). Plasa protein binding o tricyclic antidepressants in an. Biocheical Pharacology, 18, Haer, W. M. and Brodie, B. B. (1967). Application o isotope derivative technique to assay o secondary aines: Estiation o desipraine by acetylation with H3-acetic anhydride. Journal o Pharacology and Experient Therapeutics, 1, Haer, W., Idestro, C.-M. and Sjoqvist, F. (1967). Cheical control o antidepressant drug therapy. In Antidepressant Drugs, pp Excerpta Medica, International Congress Series, No. 11, Asterda. Haer, W. and Sjoqvist, F. (1967). Plasa levels o onoethylated tricyclic antidepressants during treatent with iipraine-like copounds. Lie Sciences, 6, Hurwitz, N. (1969). Predisposing actors in adverse reactions to drugs. British MedicalJournal, 1, Sjoqvist, F., Haer, W., Idestro, C.-M., Lind, M., Tuck, D., and Asberg, M. (1968). Plasa level o onoethylated tricyclic antidepressants and side-eects in an. Excerpta Medica, International Congress Series, No. 145, -7. Sjoqvist, F., Haer, W., Borgh, O., and Azarno, D. L. (1969). Pharacological signiicance o the plasa level o onoethylated tricyclic antidepressants. Excerpta Medica, International Congress Series, No. 180, J Med Genet: irst published as /jg on 1 June Downloaded ro on 13 Noveber 018 by guest. Protected by